Quick PCR ques.

Warren Gallin wgallin at gpu.srv.ualberta.ca
Wed Aug 13 19:37:51 EST 1997


In Article <v01530500b01885defb61@[129.127.90.149]>,
adobrovic at medicine.adelaide.edu.au (Alex Dobrovic) wrote:
>From: dcarpio at mail1.sas.upenn.edu (David Carpio)
>
>>I'm having a few problems with my PCR protocol, and I've heard that
>>having *too many* dNTPs can inhibit the reaction.  I was wondering what the
>>reason for that would be.
>>
>>Any thoughts?
>>
>>Dave
>
>dNTPs bind Magnesium stoiochiometrically. Taq polymerase needs free Mg++ as
>a cofactor. Typically with 0.m8M dNTPs and 1.5 mM, there is 0.7mM free
>Mg++. (actually a bit less as the DNA binds some too). If you were to
>double the dNTPs, there would be no free Mg++.
>

Although true, not the complete story.  Taq is inhibitted by concentrations
of dNTPs over 0.2 mM final concnetration, independent of magnesium
concentration.  I don't know the mechanism, but increasing dNTP
concentration above that level relaly kills the yield.

Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton,  Alberta     T6G 2E9
Canada
wgallin at gpu.srv.ualberta.ca



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